Biological tissue that is prepared via cell culture on a culture support such as collagen has low cell density. Accordingly, such biological tissue is not suitable as transplant tissue for medical purposes. A technique for preparing tissue suitable for transplantation, such as a cell sheet with high cell density, can be said to be an important technique in the tissue engineering field. However, existing methods for preparing biological tissue with high cell density have several drawbacks.
In general, sowing of cells on a culture support to form a cell sheet is extensively carried out. In order to facilitate detachment of a cell sheet, a technique of providing a layer of a temperature-responsive polymer compound on a cell adhesion surface to accelerate cell detachment has been developed, and a cell sheet that is substantially free from foreign matter can be collected. A cell sheet formed via such technique, however, is generally composed of a single layer or three or fewer cell layers. Accordingly, it is necessary to superpose a plurality of cell sheets in order to form a multilayer structure. Since a cell sheet is very thin and difficult to handle, superposition of a plurality of such cell sheets is laborious rather than easy.
Patent Document 1 and Non-Patent Document 1 each disclose a method for easily preparing a multilayered cell sheet in which cells are allowed to support magnetic fine particles thereon, magnetized cells are sowed in a culture vessel having a non-cell-adhesive bottom surface, the magnetized cells are affixed to the bottom surface with a magnetic force, cell culture is conducted to form tissue, and tissue is collected via removal of the magnetic force at the end. This technique enables preparation of multilayered cells without the need for superposition of cell sheets. With this technique, however, contamination of biological tissue with foreign matter (i.e., magnetic fine particles) cannot be avoided. Thus, such technique cannot be said to be preferable for preparation of transplant tissue for medical purposes.
Meanwhile, a method in which cells are exposed to centrifugal force during cell culture had been employed in the past for the purpose of imposing stimuli to cultured cells. For example, Patent Document 2 discloses a method for stimulating cells by regulating the dynamic environment with the application of hydrostatic pressure by centrifugal force under cell culture conditions for the purpose of suppressing cell dedifferentiation. Patent Document 2, however, does not refer to any method for detaching formed tissue. Also, what is disclosed in Patent Document 2 is a method in which centrifugal force is intermittently applied while growing cells over the period of several weeks. With such method, a tissue mass of weakly adhering spherical cells, which are referred to as spheroids, is formed (Patent Document 2, FIG. 6), and, thus, tissue that can be used as a graft, such as a cell sheet, cannot be prepared.
Patent Document 3 discloses a method for preparing a three-dimensional construct from bone cells or the like. According to the method of Patent Document 3, a cell suspension is allowed to stand in a given cloning ring, it is allowed to precipitate once, the cell suspension is allowed to stand further, tissue is formed from the precipitated cells, the tissue-forming cells are subjected to rotation culture so as to enhance oxygen- and nutrition-diffusing effects, and a three-dimensional construct of bone cells or the like with a centimeter-order size is then prepared. However, such technique has drawbacks such that, for example, it takes several days to prepare tissue, target cells are limited to bone cells or the like, tissue cannot be easily detached from a culture vessel, and a conformation cannot be regulated as intended. Accordingly, biological tissue prepared by existing techniques disadvantageously contains unfavorable components, such as magnetic fine particles, and such biological tissue is not suitable for transplantation. Further, a sufficient method for preparing biological tissue of superposed cells has not yet been provided.